Hypospadias (the abnormal placement of the urethral opening) is one of the most common birth defects in the USA, affecting approximately 1 in every 140 live male births. Despite its high incidence, relatively little is known about the causes of this common disease. A few genes have been isolated that predispose the penis to hypospadias. However, genetic causes alone cannot explain a doubling in the reported rate of hypospadias in the USA between 1970-1993. Since inappropriate estrogen exposure or inhibition of androgen signaling have both been shown to induce hypospadias, its increase has been attributed to environmental factors, with environmental endocrine disruptors (EEDs) being prime candidates. Thus, understanding the interplay between hormones and the critical molecular pathways regulating urethral closure is essential to define the causes of hypospadias and potential targets for the clinical management of this disease. In this proposal we will define the function of a novel long noncoding RNA molecule (lnc353) that is necessary for urethral closure in mice. Deletion of lnc353 causes a complete failure of urethral closure resulting in a severe penoscrotal hypospadias, mirroring human severe hypospadias phenotypes. We have shown that lnc353 is flanked by multiple androgen and estrogen receptor response elements suggesting it is under direct hormonal control and a potential target of endocrine disruption. Furthermore, lnc353 is unusually highly conserved in the human genome both at the nucleotide level (>80%) and in its physical location, suggesting an important developmental function. In this proposal we will define the function of lnc353 in urethral closure, its interaction with know and novel penile patterning networks, its hormonal control and potential for endocrine disruption. In future work, we will determine if mutations in lnc353 are associated with severe hypospadias in humans. This work will characterize a critical new player in normal urethral closure and penile development. Our findings will provide much needed information on the genetic and hormonal causes of this common disease and aid in the development of new ways to diagnose and treat this condition. Such studies are fundamental before we can assess clinical management of this disease and the potential reasons for the dramatic increase in hypospadias incidence over recent decades. This research is inline with the missions of the NIH, specifically by Improving the health of the Nation by conducting and supporting research in the causes, diagnosis, prevention, and cure of human diseases; in the processes of human growth and development; and in the biological effects of environmental contaminants. PUBLIC HEALTH RELEVANCE: Hypospadias (the abnormal placement of the urethral opening on the penis) is one of the most common birth defects in the USA, affecting approximately 1 in every 140 live male births. Inappropriate exposure to hormones during development has been shown to cause hypospadias and an increase in environmental endocrine disrupting chemicals has been implicated in the doubling in incidence of this disease in the USA between 1970-1993. In this proposal, we will investigate the role of a new long noncoding RNA gene that is necessary for urethral closure and has the potential for hormonal disruption suggesting it could be cause of human sporadic hypospadias.